Regulation of renal ion transport by the CUL3-WNK-SPAK pathway

CUL3-WNK-SPAK 通路对肾离子转运的调节

• 批准号: 10083727
• 负责人: JAMES A MCCORMICK
• 金额: $ 33.88万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10083727
• 关键词: Address; Affect; Amino Acids; Animals; Autophagocytosis; Binding; Blood Pressure; Cardiac Myocytes; Cells; Chronic Kidney Failure; Complex; Cullin Proteins; Cultured Cells; Data; Defect; Development; Dietary Potassium; Disease; Disputes; Distal; Distal convoluted renal tubule structure; Equilibrium; Exons; Familial disease; Functional disorder; Gitelman syndrome; Goals; Heart failure; Heterozygote; Homeostasis; Human; Hypertension; Hypokalemia; Immunofluorescence Immunologic; In Vitro; Ion Transport; Kidney; Knock-out; Knockout Mice; Knowledge; Lead; Ligase; Limb structure; Maintenance; Mediating; Messenger RNA; Modeling; Molecular; Mus; Mutation; Nephrons; Neurons; Normal Range; Pathway interactions; Phenotype; Phosphotransferases; Physiology; Plasma; Play; Polyuria; Potassium; Primary Cell Cultures; Progress Reports; Proteins; Rare Diseases; Regulation; Role; SLC11A2 gene; Scaffolding Protein; Skeletal Muscle; Sodium; System; Testing; Thick; Ubiquitination; Work; base; blood pressure regulation; cullin-3; driving force; exon skipping; extracellular; familial hyperkalemic hypertension; gain of function; hyperkalemia; in vivo; inhibitor/antagonist; insight; loss of function; meter; mouse model; mutant; novel therapeutics; renal epithelium; scaffold; sensor; ubiquitin ligase

Project Summary The kidney plays a key role in maintaining plasma [K+], with distal segments of the nephron fine-tuning K+ secretion to keep it in the normal range. We previously proposed that the renal distal convoluted tubule (DCT) plays a key role by sensing plasma [K+]. Decreasing plasma [K+] by dietary K+ restriction activates the WNK- SPAK/OSR1-NCC pathway, and increased NaCl reabsorption though NCC reduces delivery of sodium to, and possibly remodels, distal K+ secreting segments to lower K+ secretion. The disease Familial Hyperkalemic Hypertension (FHHt) is caused by increased NCC activation due to mutations in WNKs, Cullin 3 (CUL3), and KLHL3. The Cullin Ring Ligase (CRL) complex, composed of the scaffold CUL3, the substrate adaptor KLHL3, and the ligase RING, degrades WNKs. The effects of mutant CUL3, produced by skipping of exon 9 which causes internal deletion of 57 amino acids (CUL3-∆9), are controversial. CUL3-∆9 triggers its own degradation in vitro, and also in a mouse model of CUL3 FHHt. Thus, the prevailing model is that CUL3-∆9 causes FHHt by inducing CUL3 haploinsufficiency. Our preliminary data in CUL3 heterozygote mice and a new mouse model of CUL3-∆9 FHHt do not support this, and we hypothesize that CUL3-∆9 exerts dominant effects to cause FHHt and dysregulate the plasma [K+] sensor. We propose that CUL3-∆9 causes FHHt by a combined effect of lowering abundance of itself and of KLHL3. Our data suggest that NKCC2 activation along the thick ascending limb (TAL) may also contribute to FHHt. Finally, we previously generated kidney-specific CUL3 knockout (KO) mice, and found that they display a severe phenotype (polyuria and chronic kidney disease), with defects along multiple nephron segments. Our overall aim is to determine the mechanisms underlying CUL3-∆9-mediated FHHt, and gain insight into CUL3 function in the kidney. In Aim 1 we will determine the effects of CUL3-∆9 expression and CUL3 KO specifically along DCT to determine whether CRL disruption along DCT is sufficient to cause FHHt. We will determine the effects of CRL disruption on KLHL3 in mice, since we found CUL3-∆9 inappropriately degrades it in cultured cells. We will also directly test whether mice with lower abundance of CUL3 and KLHL3 develop FHHt. In Aim 2 we will determine whether remodeling of K+-secreting segments occurs in FHHt mediated by CUL3-∆9, and examine the effects of CRL disruption on NKCC2 activity. Some models suggest that CUL3-∆9 leads to dramatically lower CRL activity, but data suggest this would be lethal. We propose that CUL3-∆9 may exert unique effects that cause it to preferentially degrade certain CRL adaptors. Therefore, in Aim 3 we will examine effects of CUL3-∆9 on other CRL adaptors and substrates in our mouse models and in primary cell culture.

项目摘要 肾脏在维持血浆[K+]方面起着关键作用，并具有肾小管微调K+的远端段 将其保持在正常范围内的分泌。我们先前提出了肾远端曲折管（DCT） 通过感测等离子体[K+]发挥关键作用。饮食K+限制减少血浆[K+]会激活WNK- SPAK/OSR1-NCC途径，尽管NCC降低了钠的递送，并增加了NaCl的重吸收 可能会重塑，远端K+分泌段以降低K+分泌。疾病家族性高钾血症 高血压（FHHT）是由于WNK，Cullin 3（Cul3）和 Klhl3。 Cullin环连接酶（CRL）复合物，由支架Cul3，底物适配器KLHL3组成 和连接酶环，降解了wnks。突变cul3的效果，通过跳过外显子9产生的效果 引起57个氨基酸（CUL3-∆9）的内部缺失是有争议的。 Cul3-∆9触发自己的退化 体外，以及在Cul3 FHHT的小鼠模型中。那是普遍的模型，即Cul3-∆9导致FHHT 诱导CUL3单倍宽度。我们在Cul3杂合小鼠中的初步数据和新的鼠标模型 Cul3-∆9 FHHT不支持这 并使血浆[K+]传感器失调。我们建议CUL3-∆9通过 降低自身和KLHL3的抽象。我们的数据表明NKCC2沿厚升的激活 肢体（TAL）也可能有助于FHHT。最后，我们以前产生了肾脏特异性的Cul3敲除（KO） 小鼠，发现它们表现出严重的表型（多尿和慢性肾脏疾病），存在缺陷 多个肾单位段。我们的总体目的是确定CUL3-∆9介导的机制 FHHT，并深入了解肾脏中的Cul3功能。在AIM 1中，我们将确定CUL3-∆9的影响 表达和CUL3 KO专门沿DCT确定沿DCT的CRL破坏是否足够 引起FHHT。我们将确定CRL破坏对小鼠KLHL3的影响，因为我们发现CUL3-∆9 不适当地将其降解在培养的细胞中。我们还将直接测试较低丰度的小鼠 CUL3和KLHL3开发FHHT。在AIM 2中，我们将确定是否重塑K+分配段 发生在由CUL3-∆9介导的FHHT中，并检查CRL破坏对NKCC2活性的影响。一些 模型表明CUL3-∆9导致CRL活性大大降低，但数据表明这将是致命的。 我们建议CUL3-∆9可能会执行独特的效果，从而使其优先降解某些CRL 适配器。因此，在AIM 3中，我们将检查Cul3-∆9对我们的其他CRL适配器和底物的影响 小鼠模型和原发性细胞培养。